Article pubs.acs.org/EF
Influence of Carbon and Nitrogen Nutrient Supplementations on Biodegradation of Lignin by Interkingdom Fusants Yuancai Lv,† Yuancai Chen,*,†,‡ Youyou Sun,† and Yongyou Hu†,‡ †
State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Science, South China University of Technology, Guangzhou, Guangdong 510640, People’s Republic of China ‡ Ministry of Education Key Laboratory of Pollution Control and Ecological Remediation for Industrial Agglomeration Area, College of Environment and Energy, South China University of Technology, Guangzhou, Guangdong 510006, People’s Republic of China ABSTRACT: Lignin degradation by three interkingdom fusants (PE-6, PE-7, and PE-9) of Psathyrella candolleana and Enterobacter cloacae were investigated. The influence of nitrogen and carbon supplementations on the growth, lignin degradation, and laccase activity of fusants was also discussed. The results showed that NH4Cl and sucrose were the best for nitrogen and carbon supplementations, respectively. PE-9 had the highest lignin degradation efficiency (46.3 ± 1.2%) and laccase activity (16.0 ± 0.4 units/g of cell). Moreover, the growth kinetics of the fusants was studied by a logistic model. The correlation coefficient R2 values of three fusants were 0.999, 0.997, and 0.998, respectively. PE-6 had the maximal CM value of 2.04, and PE-9 had the largest k value of 0.505. Finally, the Andrews model was employed to analyze the lignin degradation kinetics. The results indicated that lignin degradation would be inhibited when the lignin concentration reached 120−160 mg/L. PE-9 was less sensitive to substrate inhibition in lignin degradation with the largest qmax, Ks, and Ki values of 1.88 mg L−1 h−1, 52.8 mg/L, and 516.4 mg/L, respectively. study.10 Our group also found that the fusants, obtained by IKPF, had excellent capacity of degrading lignin and pentachlorophenol.12,13 Clearly, constructing more functional strains, which had high degradation efficiency and maximized environmental adaptability, was an important issue. In this experiment, three interkingdom fusants (named PE-6, PE-7, and PE-9), which were obtained by interkingdom protoplast fusion14−16 of Psathyrella candolleana and Enterobacter cloaca, were selected to study their capacities for degrading lignin. An enzyme assay indicated that the fusants could produce laccase in alkalescence conditions (pH 7−9), which was significant in the treatment of alkalescence pulping wastewater. To understand well the characteristics of the fusants, the growth kinetics and the lignin degradation capability will be studied. Furthermore, the effects of nitrogen and carbon sources and the degradation kinetics will also be discussed.
1. INTRODUCTION The pulp and paper wastewater is regarded as a significant pollution source because of its high levels of chemical oxygen demand (COD), low biodegradability, and strong toxicity. Abundant lignin in the wastewater is resistant to degradation by most treatment microorganisms and, therefore, cannot be treated effectively. White-rot1−3 and brown-rot4,5 fungi are wellknown for their lignin degradation capability and functional extracellular oxidative enzymes. However, the pH needs to be maintained at 4−5 to have good treatment performance. The pH of the wastewater is commonly at 8−9, which greatly reduces practicality of using the fungi for treating lignin. Although other bacteria have demonstrated their capabilities of degrading and assimilating lignin6,7 because of their environmental adaptability and biochemical versatility, the degradation efficiency is not satisfactory. Therefore, there is an urgent need for innovative biological alternatives to treat pulp and paper wastewater effectively and economically. As a new cell engineering technology, protoplast fusion technology has been used in many fields, such as genetics, immunology, food science, and environmental protection. In the field of wastewater treatment, interkingdom protoplast fusion (IKPF) technology provided a potential to build an engineering bacteria with high degradation capability, specific degradation capability, or high adaptation capability using eukaryotes and prokaryotes. Cheng’s group constructed two functional strains (Fhhh8 and Xhhh9) through IKPF technology. The two fusants simultaneously inherited high capacities of degradation, flocculation, and adaptation from its three parental strains.10 The experimental results of biodegradation kinetics demonstrated that the functional strains were characterized with high biodegradation capacity of pharmaceutical and petrochemical,11 which was further confirmed by a pilot © 2014 American Chemical Society
2. MATERIALS AND METHODS 2.1. Materials. PE-6, PE-7, and PE-9 were three interkingdom fusants, which were obtained by interkingdom protoplast fusion of P. candolleana and E. cloacae. The lignin (typical Mn of 5000 and typical Mw of 28 000) was purchased from Sigma. The nutrient medium contained 10 g/L glucose and mineral salt medium (MSM) at pH 7. The compositions of MSM were KH2PO4 (0.42 g/L), K2HPO4 (0.375 g/L), (NH4)2SO4 (0.244 g/L), NaCl (0.015 g/L), CaCl2·2H2O (0.015 g/L), MgSO4·7H2O (0.05 g/L), FeCl3·6H2O (0.054 g/L), MnSO4·H2O (0.0025 g/L), ZnSO4·7H2O (0.002 g/L), CoCl2·6H2O (0.003 g/L), CuSO4·5H2O (0.006 g/L), and Na2BO3 (0.003 g/L). A phosphate buffer (pH 7) was prepared by Received: January 16, 2014 Revised: June 24, 2014 Published: June 25, 2014 4699
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Laccase activity was determined by an UV/vis spectrophotometer.18 Laccase activity was estimated by monitoring the A420 increase related to the rate of oxidation of 0.5 mM 2,2′-azino-bis(3-ethylthiazoline-6sulfonate) (ABTS) (ε420 = 36 000 M−1 cm−1) in 0.1 M sodium acetate buffer (pH 5.0) at 25 °C. The reaction mixture (Vtot =1 mL) contained buffer, ABTS, and sample. Laccase activity of 1 unit is defined as the amount of enzyme that transforms 1 μmol of ABTS/min. 2.7. Kinetics Model. The model of logistic was chosen to describe the growth kinetics of fusants, which was defined as
dissolving 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L K2HPO4, and 0.2 g/L KH2PO4 in deionized water (Millipore, Milli-Q). Prior to use, the MSM and phosphate buffer were sterilized in an autoclave at 121 °C for 30 min. The lignin substrate solution was prepared by dissolving lignin in MSM to the required concentrations. 2.2. Isolation of Parental Protoplasts. The isolation of parental protoplasts was described in the study by Chen et al.12 Briefly, E. cloacae cells were harvested in a 5 mL tube by centrifugation at 3000 rpm and 5 °C for 10 min, washed 3 times with phosphate-buffered saline (PBS) buffer, and resuspended in 1 mL of NSM buffer (0.55 mol/L NaCl, 0.2 mol/L sodium succinate, and 0.02 mol/L MgCl2· 6H2O at pH 6.8) containing 1 mL lysozyme solution (2000 U/mL) [0.1 g of lysozyme powder (1000 units/mg) was dissolved in 50 mL of NSM solution and filtered with a 0.22 μm filter] and 1 mL of ethylenediaminetetraacetic acid (EDTA) solution (0.13 mol/L Na2EDTA and 0.55 mol/L NaCl). The cells were shaken at 150 rpm at 37 °C for 60 min to allow for the digestion of the peptidoglycan layer. The mycelium of P. candolleana was harvested in a 5 mL tube by centrifugation at 3000 rpm and 5 °C for 10 min, washed 3 times with PBS buffer, and resuspended in 1 mL of NSM buffer containing 1 mL of mixed enzyme solution [1% cellulose (1000 units/mL) and 0.2% helicase (200 units/mL), two enzymes dissolved in 0.6 mol/L MgSO4 solution and filtered with a 0.22 μm filter] and 1 mL of EDTA solution. The mycelium was shaken at 150 rpm at 37 °C for 60 min to remove the cell wall of P. candolleana. 2.3. Protoplast Fusion. Protoplast fusion was performed according to an established process,12 with some modification. The prepared parental protoplast suspensions were adjusted to approximately the same turbidity and mixed at a volume ratio of 1:1. Mixed protoplasts were harvested by centrifugation at 2000 rpm for 10 min at 5 °C and then washed twice with 2 mL of NSM buffer. Thereafter, 1 mL of 30% PEG6000 (30% PEG6000, Sigma Chemicals, St. Louis, MO, prepared in STC buffer containing 0.6 M sorbitol, 10 mM Tris− HCl, and 10 mM CaCl2 at pH 6.5) was added. The fusion mixture was incubated at 37 °C for 5 min and then diluted with 1 mL of NSM buffer. After washed twice with NSM buffer, the fusants were obtained by centrifugation at 2000 rpm for 10 min. 2.4. Free Suspension Cultivation. Two rings of three fusants were inoculated into the nutrient medium (250 mL) in 500 mL Erlenmeyer flasks from the solid media. After inoculation, the flask was capped with cotton plugs and placed in a shaker controlled at 150 rpm and 35 °C. The samples were withdrawn at suitable time intervals, and the cell concentration was measured. 2.5. Biodegradation Experiments. When the OD600 value of the precultured cells reached 0.1−0.2, 5 mL of bacterial suspension was inoculated in a 250 mL Erlenmeyer flask. Then, 100 mL of the solution containing MSM and lignin was poured to give an initial concentration of 200 mg/L. The pH of the solution was adjusted to 9.0 using sodium hydroxide solution (10 wt %). The cells were cultivated at 35 °C and 150 rpm. The samples were withdrawn at suitable time intervals, and the cell density and concentrations of lignin were measured. 2.6. Analysis of Cells, Lignin, and Enzyme Activity. The cell concentration in the sample was analyzed by measuring the optical density (OD) at 600 nm using an ultraviolet/visible (UV/vis) spectrophotometer (Jasco UV-550, Japan) with the culture medium as a reference. The samples exceeding 0.8 of OD600 were appropriately diluted with the culture medium, so that the Beer−Lambert law was applied. The concentrations of lignin were analyzed by the acetyl bromide spectrophotometric method at 280 nm,17 which is treated as follows: (1) the samples in the 10 mL centrifuge tube were first treated with freeze-drying; (2) 0.5 mL of 25% acetyl bromide in glacial acetic acid (HAcBr) was added to each tube; (3) all of the tubes were capped and put in a 50 °C water bath for 30 min; and (4) after cooling the samples, all tubes received 2.5 mL of acetic acid (HAc), 1.5 mL of 0.3 M NaOH, and 0.5 mL of 0.5 M hydroxylamine hydrochloride solution.
Cx(t ) = CM /[1 + (C M /C0 − 1)e−kt ]
(1)
where Cx is the absorbance unit at 600 nm (OD), CM is the maximal absorbance unit at 600 nm (OD), C0 is the initial absorbance unit at 600 nm (OD), k is the kinetic constant (h−1), and t is the time (h). At the same time, the model of Andrews was chosen to analyze the lignin degradation kinetics. The model can be described as follows: q = qmax S /(S + K s + S2/K i)
(2) −1
−1
where q is the substrate concentration (mg L h ), qmax is the maximum degradation rate (mg L−1 h−1), Ks is the substrate-affinity constant (mg/L), and Ki is the substrate-inhibition constant (mg/L). 2.8. Statistical Analysis. All experiments were carried out in triplicate. The data were expressed as the mean ± standard deviation. The standard deviations for all measurements ranged from 1.5 to 9.8%. The difference between the lines in the figures has been analyzed by a t test (Minitab 16 software) based on a 5% level of significance.
3. RESULTS AND DISCUSSION 3.1. Effect of Different Nitrogen Supplementations on the Growth, Lignin Degradation, and Laccase Activity. In this work, 0.5 g/L of four nitrogen supplementations [NH4Cl, (NH4)2SO4, NH4NO3, and (NH4)2C2O4] was added to the medium, in which lignin (200 mg/L) was the sole carbon source. Figure 1 showed the growth curves of three fusants under four kinds of nitrogen supplementations. Evidently, three growth curves were similar, and the cells increased rapidly within 5 days but slowly after 5 days. The OD600 value increased from 0.25 to 1.50 approximately over the course of 6 days. No obvious increase of the OD600 value was detected in the control (without nitrogen supplementation). In comparison to (NH4)2C2O4, the effects of NH4Cl, (NH4)2SO4, and NH4NO3 were rather remarkable; their OD600 values could reach 1.0−1.4 approximately. The OD600 value of (NH4)2C2O4 only reached 0.7−0.8 approximately. It revealed that NH4Cl, (NH4)2SO4, and NH4NO3 were better for nitrogen supplementation, among which NH4Cl was the best for nitrogen supplementation. For PE-7 and PE-9, the effects on the growth of four nitrogen supplementations was NH4Cl > (NH4)2SO4 > NH4NO3 > (NH4)2C2O4. However, for PE-6, it was NH4Cl > NH4NO3 > (NH4)2SO4 > (NH4)2C2O4. Nitrogen was an essential nutriment for microbial metabolism and also played a vital role in laccase production.19,20 Table 1 listed lignin degradation efficiency (after 2 days) and laccase activity of the fusants under different nitrogen supplementations. The results illustrated that the lignin degradation by three fusants was consistent with their growth. NH4Cl was still the best for nitrogen supplementation in lignin degradation. The lignin degradation efficiencies of PE-6, PE-7, and PE-9 were 35.3 ± 0.9, 30.7 ± 0.9, and 36.6 ± 0.5%, respectively. A previous study had proven that a certain level of nitrogen supplementation, such as NH4NO3, L-asparagine, and ammonium tartrate, could promote the degradation of lignin by P. chrysosporium.21 However, the laccase activities of the fusants were a little different with their growth. NH4Cl, (NH4)2SO4, and NH4NO3 4700
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respectively) were lower than the control (4.5 ± 0.2, 2.6 ± 0.2, and 4.8 ± 0.3 units/g of cell, respectively), revealing that the laccase activity might be inhibited by (NH4)2C2O4. 3.2. Effects of Different Carbon Supplementations on the Growth, Lignin Degradation, and Laccase Activity. To study the effects of carbon supplementation on cell growth and laccase activity, 1.0 g/L sucrose, glucose, starch, and lactose was added to the medium, which contained 0.5 g/L NH4Cl and 200 mg/L lignin. It can be seen that sucrose and glucose had an obvious effect on the growth of the fusants (Figure 2). For PE9, the OD600 value reached the maximum (1.63) after 1 day and stabilized at 1.10 thereafter 5 days. However, the effects of starch and lactose on the cell growth were unconspicuous with the control (without extra carbon sources). It can also be seen in Table 2 that the lignin degradation and laccase activity of three fusants were both consistent with their growth. The effect of four carbon sources was sucrose > glucose > starch > lactose. However, the difference of lignin degradation between sucrose and glucose was less obvious. The lignin degradation efficiencies of PE-6, PE-7, and PE-9 with sucrose and glucose were 41.7 ± 0.9, 36.2 ± 0.6, and 46.3 ± 1.2% and 38.4 ± 0.8, 31.8 ± 0.6, and 42.3 ± 0.5%, respectively. Meanwhile, the laccase activities of PE-6, PE-7, and PE-9 with sucrose were 15.3 ± 0.2, 14.1 ± 0.4, and 16.0 ± 0.4 units/g of cell, respectively, which were obviously higher than the control (8.0 ± 0.2 units/g of cell). These results suggested that different carbon sources had a significant impact on laccase activity, which was in accordance with reports by Jaouani et al.22 and Shraddha et al.19 Laccase was well-known for its remarkable capability of degrading lignin in wood, which was produced by white-rot basidiomycetes. The addition of an extra carbon source promoted the growth of fusants, because they needed an additional readily metabolizable carbon source for growth. Thus, abundant laccase was induced, resulting in effective lignin degradation. In the study by Wu et al., it was found that glucose could promote the lignin degradation by Pleurotus ostreatus and the degradation efficiency could reach 68% on day 10 at 1 g/L glucose.20 Considering the growth and laccase activity, sucrose was chosen as the optimal extra carbon source in our experiment. Additionally, a comparison of the fusants and their parent strains on lignin degradation and laccase activity was studied under the conditions of 1.0 g/L sucrose and 0.5 g/L NH4Cl at pH 9.0. The laccase activity and lignin concentrations were analyzed after 2 days of incubation. In the study, the fusants demonstrated higher capabilities for degrading lignin and higher laccase activity than their parent strains at pH 9.0 (Figure 3). Moreover, the fusant PE-9 showed the highest lignin degradation efficiency (46.3 ± 1.2%) and highest laccase activity (16.0 ± 0.4 units/g of cell). However, one of the parent strains, E. cloacae (E), almost had no capability of degrading
Figure 1. Effects of different nitrogen sources on the growth of the fusants: (a) PE-6, (b) PE-7, and (c) PE-9.
could improve the laccase activity. The laccase activity of PE-9 reached the highest (8.0 ± 0.3 units/g of cell) in the medium with NH4Cl, while the laccase activities of three fusants with (NH4)2C2O4 (3.2 ± 0.3, 1.9 ± 0.1, and 3.5 ± 0.2 units/g of cell,
Table 1. Effects of Different Nitrogen Sources on Lignin Degradation and Laccase Activitya PE-6 nitrogen source NH4NO3 (NH4)2SO4 NH4Cl (NH4)2C2O4 control a
LDE (%) 28.6 26.2 35.3 14.2 8.3
± ± ± ± ±
0.8 0.5 0.9 0.6 0.7
PE-7
LA (units/g of cell) 6.1 5.1 7.7 3.2 4.5
± ± ± ± ±
0.3 0.4 0.2 0.3 0.2
LDE (%) 27.7 29.1 30.7 7.4 5.6
± ± ± ± ±
1.2 0.8 0.9 0.7 0.5
PE-9
LA (units/g of cell) 5.4 7.0 6.4 1.9 2.6
± ± ± ± ±
0.3 0.1 0.2 0.1 0.2
LDE (%) 25.9 27.5 36.6 18.8 10.2
± ± ± ± ±
0.9 0.6 0.5 0.7 0.4
LA (units/g of cell) 4.5 5.4 8.0 3.5 4.8
± ± ± ± ±
0.2 0.4 0.3 0.2 0.3
LDE, lignin degradation efficiency; LA, laccase activity. 4701
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Figure 3. Lignin degradation efficiency and laccase activity of PC, E, PE-6, PE-7, and PE-9.
that the fusants had better adaptability than PC. Sivakumar et al. also found that fusion recombinant F4 (0.551 unit/mL) showed higher laccase activity than its parent strain Streptomyces sp. (0.279 unit/mL) after 5 days of incubation.15 The fusion with eukaryotes resulted in low laccase activity of the fusants, but they still kept excellent capabilities of lignin degradation. On the one hand, the fusants only inherited partial genes of their parent, PC, resulting in lower laccase activity than PC. On the other hand, the fusants also inherited the characteristics of another parent, E, and they had high adaptation capability and reproductive capacity. The fusants could quickly reach stationary phase in 24 h under alkalescence conditions, which grew much more quickly than PC. Thus, the amount of laccase produced by fusants was greatly larger than PC, causing high lignin degradation. In the study by McCarthy and Broda, only 8% 14C-labeled lignin in wheat straw was degraded to 14CO2 after 14 days with both Streptomyces and white-rot fungi.23 As reported by Kern, a Xanthomonas strain just mineralized about 30% 14C-labeled synthetic lignin in 20 days.24 Thus, the fusants had an alluring prospect in the lignin degradation. 3.3. Growth Kinetics of Fusants. The logistic model was selected to analyze the growth experiment. It is evident that the growth curves of three fusants (Figure 4) were similar. The curves clearly showed three phases of the growth of the cells: lag phase (0−4 h), logarithmic phase (5−18 h), and stationary phase (after 18 h). In comparison to one of their parent strains, E, whose stationary phase of growth curve appeared only after 8 h, the proliferation rate of fusants was lower. However, it was rather higher than another parent strain, PC (not shown in Figure 4). Table 3 listed growth kinetics parameters of three fusants, which are obtained by a nonlinear least-squares regression method using the Origin software. It is noticed that the high correlation coefficient R2 values (0.999, 0.997, and
Figure 2. Effects of different carbon sources on the growth of the fusants: (a) PE-6, (b) PE-7, and (c) PE-9.
lignin and producing laccase. The lignin degradation efficiency and laccase activity of P. candolleana (PC) were also rather unsatisfactory in alkalescent conditions. The results indicated
Table 2. Effect of Different Carbon Sources on Lignin Degradation and Laccase Activitya PE-6 carbon source glucose lactose starch sucrose control a
LDE (%) 38.4 33.7 36.5 41.7 31.5
± ± ± ± ±
0.8 1.0 0.7 0.9 0.6
PE-7
LA (units/g of cell) 12.8 9.3 11.5 15.3 8.3
± ± ± ± ±
0.3 0.5 0.4 0.2 0.2
LDE (%) 31.8 27.6 29.0 36.2 26.3
± ± ± ± ±
0.6 0.9 0.5 0.6 0.7
PE-9
LA (units/g of cell) 11.1 7.7 9.9 14.1 7.0
± ± ± ± ±
0.3 0.3 0.2 0.4 0.2
LDE (%) 42.3 32.4 37.9 46.3 35.4
± ± ± ± ±
0.5 0.9 0.9 1.2 0.6
LA (units/g of cell) 15.0 8.9 12.1 16.0 8.0
± ± ± ± ±
0.5 0.3 0.4 0.4 0.2
LDE, lignin degradation efficiency; LA, laccase activity. 4702
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Figure 4. Growth of three fusants and E. cloacae and kinetics fit of the growth of fusants using the logistic model.
Figure 5. Kinetics fit of lignin degradation by fusants using the Andrews model.
0.998) of three logistic equations suggested that the logistic model could well describe the growth of fusants. Among the CM values of the fusants obtained in this work, the CM value of PE6 (2.04) was the highest, followed by PE-9 (1.95), with the lowest CM value (1.61) occurring for PE-7, which revealed that PE-6 had better adaptability in the medium that PE-9 and PE-7. The kinetic constant k values of three fusants were 0.416, 0.391, and 0.505, respectively, and the largest k value of PE-9 reflected its largest growth rate in the medium. 3.4. Degradation Kinetics of Lignin. The Andrews model was selected for the degradation kinetics analysis. The curves (Figure 5) illustrated that, as the lignin concentration increased, the degradation rates of PE-6, PE-7, and PE-9 first reached the maximum (1.884, 1.493, and 1.972 mg L−1 h−1, respectively) and then decreased when the lignin concentration reached 120−160 mg/L, revealing substrate inhibition in lignin degradation. Zhao et al. found that the laccase activity was inhibited when the concentration of cotton pulp black liquor increased between 600 and 700 mg/L, resulting in unsatisfactory lignin degradation by P. ostreatus.25 With analysis by a nonlinear least-squares regression method using the Origin software 8.5, the kinetics equations and their parameters were obtained (Table 4). Additionally, the correlation coefficient R2 values of three fitting equations were 0.985, 0.949, and 0.986, respectively, suggesting a satisfactory description by the Andrews model. In the case of lignin degradation, Zhao et al. obtained the values of qmax (1.46 mg L−1 h−1), Ks (36.7 mg/L), and Ki of (308.3 mg/L) in lignin degradation by P. ostreatus.25 For PE-6 and PE-9, the values of qmax (1.80 and 1.88 mg L−1 h−1), Ks (51.6 and 52.8 mg/L), and Ki (491.1 and 516.4 mg/L) were all larger than P. ostreatus, indicating their better capability of lignin degradation. The qmax of PE-7 (1.42 mg L−1 h−1) was lower than that of P. ostreatus, but the values of Ks (41.4 mg/L) and Ki (419.0 mg/L) were larger than those of P. ostreatus. Moreover, the largest qmax (1.88 mg L−1 h−1) of PE-9 suggested that lignin degraded by PE-9 more rapidly than PE-6 and PE-7. In comparison to the Ks values of PE-6 (51.6 mg/L) and PE-7 (41.4 mg/L), PE-9 (52.8 mg/L) had a slightly stronger affinity to lignin. Meanwhile, lthe argest Ki of 516.4 mg/L revealed that
PE-9 was least sensitive to substrate inhibition in lignin degradation. However, the typical wastewater was a very complex system, in which there were other carbon and nitrogen nutrient supplementations and chemicals. Part of the chemicals might be toxic and inhibit the metabolism of the microorganisms, resulting in some difference of the kinetic relationships between the synthetic wastewater in our laboratory and the typical wastewater. Therefore, the kinetic relationships in the typical wastewater needed to be further studied, but the results in the paper could be considered as a reference.
4. CONCLUSION Three interkingdom fusants (PE-6, PE-7, and PE-9) inherited simultaneously the high capacities of lignin degradation, laccase production, and environmental adaptation from their parental strains E. cloacae and P. candolleana. The fusants also demonstrated better efficiency on lignin degradation than P. candolleana at pH 9.0, which was significant in the treatment of neutral or alkalescence pulping wastewater. Different nitrogen and carbon supplementations both had remarkable influence on the growth, lignin degradation, and laccase activity of fusants. The results showed that NH4Cl and sucrose were the best for nitrogen and carbon supplementations, respectively. PE-9 had the highest lignin degradation efficiency (46.3 ± 1.2%) and laccase activity (16.0 ± 0.4 units/g of cell). Moreover, the growth kinetics of the fusants was well-described by the logistic equation with high R2 values of 0.999, 0.997, and 0.998, respectively. PE-6 had the maximal CM value of 2.04, while PE9 had the largest k value of 0.505. Furthermore, the Andrews model could well describe the lignin degradation kinetics, and the results indicated that substrate inhibition occurred in lignin degradation once the lignin concentration reached 120−160 mg/L. PE-9 was the least sensitive to lignin inhibition in lignin degradation because of the largest qmax, Ks, and Ki values of 1.88 mg L−1 h−1, 52.8 mg/L, and 516.4 mg/L.
Table 3. Growth Kinetics Parameters of Three Fusants fusant
CM
C0
k
logistic equation
R2
PE-6 PE-7 PE-9
2.04 1.61 1.95
0.0251 0.0231 0.0136
0.416 0.391 0.505
CPE‑6(t) = 0.0251e0.416t/[1 − 0.0123(1 − e0.416t)] CPE‑7(t) = 0.0231e0.391t/[1 − 0.0143(1 − e0.391t)] CPE‑9(t) = 0.0136e0.505t/[1 − 0.00695(1 − e0.505t)]
0.999 0.997 0.998
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Table 4. Andrews Parameters of Lignin Degradation by Fusants fusant
■
qmax (mg L−1 h−1)
PE-6 PE-7 PE-9
1.80 1.42 1.88
Ks (mg/L)
Ki (mg/L)
51.6 41.4 52.8
491.1 419.0 516.4
2
q(PE‑6) = 1.80S/(S + 51.6 + S /491.1) q(PE‑7) = 1.42S/(S + 41.4 + S2/419.0) q(PE‑9) = 1.88S/(S + 52.8 + S2/516.4)
0.985 0.949 0.986
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-13672458060. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the National Natural Science Foundation of China (20977033) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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R2
Andrews equation
REFERENCES
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dx.doi.org/10.1021/ef500679r | Energy Fuels 2014, 28, 4699−4704
